Two-dimensional RI spectroscopy: Automation with pulse shaping and application to amyloid folding

Two dimensional infrared (2D IR) spectroscopy has proven itself as a unique tool for probing the structures and dynamics of chemical and biological systems. Now, 2D IR spectroscopy has progressed to the point of being a ready-to-operate method for general researchers to address novel scientific questions. For that purpose, this thesis includes three practices: inventing a technique for mid-infrared pulse shaping; developing an automated method of collecting 2D IR spectra; applying it to delineate the pathway of amyloid formation. First, we developed a means to shape femtosecond pulses in the mid-infrared using a germanium acousto-optic modulator (Ge AOM). The Ge AOM directly modulates the amplitude and phase of mid-IR light between 2-18 mum, producing intense shaped pulses with high resolution and good phase stability. Second, we combined the pulse shaper with a pump-probe geometry to develop a new method of 2D IR spectroscopy. The pump-probe geometry simplifies the optical setup and produces properly phased absorptive lineshapes without additional data processing. The pulse shaper not only reproduces most conventional methods, but also allows new pulse shapes and phase combinations to further enhance 2D IR spectroscopy. Moreover, eliminating moving parts accelerates data collection so that an entire 2D IR spectrum can be obtained in <1 sec. Such ease of use, versatility and speed of our pulse shaping 2D IR method enables more sophisticated and reliable experiments. Finally, we tracked amyloid formation of human Islet amyloid polypeptide (hIAPP), related to type II diabetes. By continuously scanning 2D IR spectra, we can follow the hIAPP fibril formation indefinitely by collecting data on-the-fly without reinitiating the aggregation for each data point. We used isotope-labeling to probe the structural evolution of six different residues along the 37-residue hIAPP peptide. By separately monitoring the six residues, we found that the peptides nucleate near the turn of the hairpin, followed by a propagation of the two parallel beta-sheets with the N-terminal beta-sheet forming twice as fast as the C-terminal sheet. The experimental approach provides a detailed view of the assembly pathway of hIAPP fibril as well as a general methodology for studying other amyloid forming peptides.